Stugy On HER2-specific Immunotoxin Based On Nanobody Combined With PE38 Toxin

In this study,an immunotoxin that can specifically kill HER2-positive breast cancer cells was constructed by natural coupling of nanobodies and PE38 toxin through genetic engineering technology.The alpaca was immunized with HER2 as an antigen,and then the antibody in the blood of the alpaca was extracted to construct a phage library.Twenty-three HER2-positive antibodies were screened by phage library.Then,10 nanobodies with the best affinity were screened by ELISA antibody-antigen binding detection.After sequencing,four nanobodies with different sequences were finally obtained.The nanobody with the best HER2 affinity was selected and constructed into the pc DNA3.1 plasmid vector,and the nanobody protein was expressed by the E.coli TOP10 high-efficiency expression system.The protein A protein purification column was used for separation and purification,and the purified antibody protein was subjected to antibody antigen binding detection and Western Blot analysis.The results proved that the nanobody protein was successfully purified.Its affinity constant K_Dwith HER2 protein is 7.2×10^-6M.The nanobody protein sequence was coupled with the PE38 toxin sequence and introduced into the p ET-30a plasmid,and carried by the E.coli BL21 high-efficiency expression system.The immunotoxin was obtained by nickel column separation and purification,and the cell killing experiment was carried out.Finally,an immunotoxin with good specific killing effect on HER2 positive cells was obtained,and the IC50 value for SKBR3 cells was791 p M.In this study,nanobody sequences with good specificity against HER2 were successfully screened;p CDNA3.1-TOP10 and p ET-30a-BL21expression systems were successfully constructed;finally,immunotoxins with good specific killing effect on HER2-positive cells were screened.This study provides a new research idea for the specific targeted therapy of HER2-positive breast cancer,which has good application prospects and no immunotoxin targeting HER2 has been marketed so far.Therefore,the immunotoxin in this study will be used in clinical treatment in the future.This success will fill a gap in the prevention and treatment of HER2-positive breast cancer.